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- PDB-7os2: Cryo-EM structure of Brr2 in complex with Jab1/MPN and C9ORF78 -

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Basic information

Entry
Database: PDB / ID: 7os2
TitleCryo-EM structure of Brr2 in complex with Jab1/MPN and C9ORF78
Components
  • Pre-mRNA-processing-splicing factor 8
  • Telomere length and silencing protein 1 homolog
  • U5 small nuclear ribonucleoprotein 200 kDa helicase
KeywordsSPLICING / mRNA Splicing / Spliceosomal Assembly / Splicing Regulation / Brr2 Helicase
Function / homology
Function and homology information


regulation of homologous chromosome segregation / cis assembly of pre-catalytic spliceosome / spliceosome conformational change to release U4 (or U4atac) and U1 (or U11) / U2-type catalytic step 1 spliceosome / RNA splicing, via transesterification reactions / mRNA cis splicing, via spliceosome / U2-type precatalytic spliceosome / U2-type catalytic step 2 spliceosome / K63-linked polyubiquitin modification-dependent protein binding / mRNA Splicing - Minor Pathway ...regulation of homologous chromosome segregation / cis assembly of pre-catalytic spliceosome / spliceosome conformational change to release U4 (or U4atac) and U1 (or U11) / U2-type catalytic step 1 spliceosome / RNA splicing, via transesterification reactions / mRNA cis splicing, via spliceosome / U2-type precatalytic spliceosome / U2-type catalytic step 2 spliceosome / K63-linked polyubiquitin modification-dependent protein binding / mRNA Splicing - Minor Pathway / chromosome, centromeric region / spliceosomal tri-snRNP complex assembly / U5 snRNA binding / U5 snRNP / U2 snRNA binding / U6 snRNA binding / pre-mRNA intronic binding / U1 snRNA binding / U4/U6 x U5 tri-snRNP complex / catalytic step 2 spliceosome / mRNA Splicing - Major Pathway / RNA splicing / helicase activity / chromosome segregation / spliceosomal complex / mRNA splicing, via spliceosome / mRNA processing / osteoblast differentiation / cellular response to tumor necrosis factor / cellular response to lipopolysaccharide / RNA helicase activity / RNA helicase / nuclear speck / ATP hydrolysis activity / RNA binding / nucleoplasm / ATP binding / membrane / identical protein binding / nucleus / cytosol
Similarity search - Function
Telomere length and silencing protein 1 / Hepatocellular carcinoma-associated antigen 59 / Brr2, N-terminal helicase PWI domain / : / N-terminal helicase PWI domain / Pre-mRNA-splicing helicase BRR2 plug domain / Sec63 Brl domain / Sec63 domain / Sec63 Brl domain / JAB1/Mov34/MPN/PAD-1 ubiquitin protease ...Telomere length and silencing protein 1 / Hepatocellular carcinoma-associated antigen 59 / Brr2, N-terminal helicase PWI domain / : / N-terminal helicase PWI domain / Pre-mRNA-splicing helicase BRR2 plug domain / Sec63 Brl domain / Sec63 domain / Sec63 Brl domain / JAB1/Mov34/MPN/PAD-1 ubiquitin protease / PROCT domain / Prp8 RNase domain IV, fingers region / PROCT (NUC072) domain / PRO8NT domain / PROCN domain / Pre-mRNA-processing-splicing factor 8, U6-snRNA-binding / Pre-mRNA-processing-splicing factor 8, U5-snRNA-binding / RNA recognition motif, spliceosomal PrP8 / PRP8 domain IV core / Pre-mRNA-processing-splicing factor 8, U5-snRNA-binding domain superfamily / Prp8 RNase domain IV, palm region / PRO8NT (NUC069), PrP8 N-terminal domain / PROCN (NUC071) domain / U6-snRNA interacting domain of PrP8 / U5-snRNA binding site 2 of PrP8 / RNA recognition motif of the spliceosomal PrP8 / PRP8 domain IV core / Pre-mRNA-processing-splicing factor 8 / JAB/MPN domain / JAB1/MPN/MOV34 metalloenzyme domain / MPN domain / MPN domain profile. / DEAD/DEAH box helicase / DEAD/DEAH box helicase domain / C2 domain superfamily / Helicase conserved C-terminal domain / helicase superfamily c-terminal domain / Immunoglobulin E-set / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / Winged helix DNA-binding domain superfamily / Ribonuclease H-like superfamily / Winged helix-like DNA-binding domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
U5 small nuclear ribonucleoprotein 200 kDa helicase / Pre-mRNA-processing-splicing factor 8 / Splicing factor C9orf78
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.76 Å
AuthorsBergfort, A. / Hilal, T. / Weber, G. / Wahl, M.C.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Research Foundation (DFG)TRR186-A15 Germany
Citation
Journal: Nucleic Acids Res / Year: 2022
Title: The intrinsically disordered TSSC4 protein acts as a helicase inhibitor, placeholder and multi-interaction coordinator during snRNP assembly and recycling.
Authors: Alexandra Bergfort / Tarek Hilal / Benno Kuropka / İbrahim Avşar Ilik / Gert Weber / Tuğçe Aktaş / Christian Freund / Markus C Wahl /
Abstract: Biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) and their recycling after splicing require numerous assembly/recycling factors whose modes of action are often poorly understood. ...Biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) and their recycling after splicing require numerous assembly/recycling factors whose modes of action are often poorly understood. The intrinsically disordered TSSC4 protein has been identified as a nuclear-localized U5 snRNP and U4/U6-U5 tri-snRNP assembly/recycling factor, but how TSSC4's intrinsic disorder supports TSSC4 functions remains unknown. Using diverse interaction assays and cryogenic electron microscopy-based structural analysis, we show that TSSC4 employs four conserved, non-contiguous regions to bind the PRPF8 Jab1/MPN domain and the SNRNP200 helicase at functionally important sites. It thereby inhibits SNRNP200 helicase activity, spatially aligns the proteins, coordinates formation of a U5 sub-module and transiently blocks premature interaction of SNRNP200 with at least three other spliceosomal factors. Guided by the structure, we designed a TSSC4 variant that lacks stable binding to the PRPF8 Jab1/MPN domain or SNRNP200 in vitro. Comparative immunoprecipitation/mass spectrometry from HEK293 nuclear extract revealed distinct interaction profiles of wild type TSSC4 and the variant deficient in PRPF8/SNRNP200 binding with snRNP proteins, other spliceosomal proteins as well as snRNP assembly/recycling factors and chaperones. Our findings elucidate molecular strategies employed by an intrinsically disordered protein to promote snRNP assembly, and suggest multiple TSSC4-dependent stages during snRNP assembly/recycling.
#1: Journal: Acta Crystallogr D Struct Biol / Year: 2019
Title: Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix.
Authors: Liebschner, D. / Afonine, P.V. / Baker, M.L. / Bunkoczi, G. / Chen, V.B. / Croll, T.I. / Hintze, B. / Hung, L.W. / Jain, S. / McCoy, A.J. / Moriarty, N.W. / Oeffner, R.D. / Poon, B.K. / ...Authors: Liebschner, D. / Afonine, P.V. / Baker, M.L. / Bunkoczi, G. / Chen, V.B. / Croll, T.I. / Hintze, B. / Hung, L.W. / Jain, S. / McCoy, A.J. / Moriarty, N.W. / Oeffner, R.D. / Poon, B.K. / Prisant, M.G. / Read, R.J. / Richardson, J.S. / Richardson, D.C. / Sammito, M.D. / Sobolev, O.V. / Stockwell, D.H. / Terwilliger, T.C. / Urzhumtsev, A.G. / Videau, L.L. / Williams, C.J. / Adams, P.D.
History
DepositionJun 7, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 23, 2022Provider: repository / Type: Initial release
Revision 1.1Mar 2, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Mar 30, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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Assembly

Deposited unit
B: U5 small nuclear ribonucleoprotein 200 kDa helicase
C: Telomere length and silencing protein 1 homolog
J: Pre-mRNA-processing-splicing factor 8


Theoretical massNumber of molelcules
Total (without water)264,7283
Polymers264,7283
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area9890 Å2
ΔGint-22 kcal/mol
Surface area83500 Å2

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Components

#1: Protein U5 small nuclear ribonucleoprotein 200 kDa helicase


Mass: 198785.797 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O75643, RNA helicase
#2: Protein Telomere length and silencing protein 1 homolog


Mass: 34081.516 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: C9orf78 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9NZ63
#3: Protein Pre-mRNA-processing-splicing factor 8


Mass: 31860.984 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PRPF8 / Production host: Escherichia coli (E. coli) / References: UniProt: Q6P2Q9

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Trimeric complex of Brr2, the Jab1/MPN domain of Prp8 and a N-terminal fragment of C9ORF78COMPLEXall0RECOMBINANT
2U5 small nuclear ribonucleoprotein 200 kDa helicase (Brr2)COMPLEX#11RECOMBINANT
3Telomere length and silencing protein 1 homolog (C9ORF78)COMPLEX#21RECOMBINANT
4Pre-mRNA-processing-splicing factor 8 (Prp8) Jab1/MPN DomainCOMPLEX#31RECOMBINANT
Molecular weight
IDEntity assembly-IDUnitsExperimental value
11KILODALTONS/NANOMETERNO
21MEGADALTONSNO
31MEGADALTONSNO
41MEGADALTONSNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Homo sapiens (human)9606
43Homo sapiens (human)9606
54Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Spodoptera frugiperda (fall armyworm)7108
22Spodoptera frugiperda (fall armyworm)7108
33Escherichia coli (E. coli)562
44Escherichia coli (E. coli)562
Buffer solutionpH: 7.6
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 120000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 40 sec. / Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5160

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameVersionCategoryDetails
2EPU2.1image acquisition
4cryoSPARC3.1CTF correction
7PHENIX1.19.2model fittingDock in Map
9PHENIX1.19.2model refinementReal space Refinement
10cryoSPARC3.1initial Euler assignment
11cryoSPARC3.1final Euler assignment
12cryoSPARC3.1classification
13cryoSPARC3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 936716
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.76 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 370493 / Symmetry type: POINT
Atomic model buildingB value: 102 / Protocol: OTHER / Space: REAL / Target criteria: CC, R.m.s.d. Bonds / Angles
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-ID
14KIT

4kit

B1
24KIT

4kit

C1
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 101.73 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.005416847
ELECTRON MICROSCOPYf_angle_d0.552122830
ELECTRON MICROSCOPYf_chiral_restr0.0442562
ELECTRON MICROSCOPYf_plane_restr0.00432933
ELECTRON MICROSCOPYf_dihedral_angle_d12.08256362

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